Project Summary The combination of pervasive multidrug-resistant bacteria and the slowing discovery of new antimicrobial therapeutics threatens to undermine many global public health advances of the past century. A main focus of the van der Donk lab is understanding the biosynthesis and mechanism of action of natural products with antimicrobial activities. In particular, the lab is interested in a class of molecules known as lanthipeptides that have potent antimicrobial activities and may be useful models for combating antibiotic resistance. Several lanthipeptides are in clinical trials to treat drug-resistant bacterial infections, and the antibiotic lanthipeptide nisin has been used for decades in the food industry to combat food-borne pathogens. Class I lanthipeptide dehydratases, generically termed LanBs, play a key role in the biosynthesis of lanthipeptides as well as other classes of natural products by converting Ser/Thr residues into dehydroalanine/dehydrobutyrine. The van der Donk lab recently demonstrated that LanBs utilize charged tRNAs to glutamylate Ser/Thr side chain alcohols, leading to an ester that is eliminated to form the dehydrated amino acid. The crystal structure of a LanB revealed a modular architecture in which the glutamylation and elimination activities are localized to separate domains. Intriguingly, many enzymes found in genome databases are missing the elimination domain entirely. These short LanB (sLanB) enzymes may thus have a different biosynthetic logic and likely produce previously unknown peptide natural products. In order to investigate these gene clusters, a research strategy aimed at determining the products and biosynthetic logic of sLanB enzymes is proposed using the latest analytical tools, including manipulation of bacterial culture conditions, transcriptional analysis, high-resolution mass spectrometry, stable isotope labeling, heterologous expression, and bioinformatics. Training in these approaches will greatly expand the skill set of the applicant. Specific goals include determining optimal culture conditions for isolation of sLanB-derived natural products in a native producer, investigating the fate of amino acid adducts derived from charged tRNAs, and establishing if sLanB enzymes in different biosynthetic contexts share a general mechanism. These aims include identifying the end products of two distinct sLanB gene clusters and evaluating their function. Given the involvement of LanB-like enzymes in a range of potent and clinically and commercially successful therapeutics, the products of sLanBs may have valuable biological activities. Additionally, understanding what reaction(s) sLanBs catalyze and initial mechanistic characterization may expand the bioinformatic toolbox for genome mining for novel natural products.